Authors’ correction
E. A. L. Fairley, J. Kendrick-Jones and J. A. Ellis (1999). The Emery-Dreifuss muscular dystrophy phenotype arises
from aberrant targeting and binding of emerin at the inner nuclear membrane. J. Cell Science 112 (15), 2571-2582.
All references to S54P or Ser54Pro are incorrect and should read as S54F or Ser54Phe, respectively.
Authors’ correction
D. Gabriel, U. Hacker, J. Köhler, A. Müller-Taubenberger, J.-M. Schwartz, M. Westphal and G. Gerisch (1999).
The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker protein.
J. Cell Science 112 (22), 3995-4005.
In Fig. 7 of this paper the numbers indicating seconds should be exchanged between panels C and E, as shown below.
Fig. 7
Authors’ correction
K. P. Williams, P. Rayhorn, G. Chi-Rosso, E. A. Garber, K. L. Strauch, G. S. B. Horan, J. O. Reilly, D. P. Baker, F. R.
Taylor, V. Koteliansky and R. B. Pepinsky (1999). Functional antagonists of sonic hedgehog reveal the importance of the
N terminus for activity. J. Cell Science 112 (23), 4405-4414.
In the discussion on page 4412, paragraph 3 line 21, the digit duplication assay was incorrectly quoted as mouse. The correct
assay is chick embryo digit duplication. In addition, line 23 should state (S. Pagan, D. P. Baker, K. P. Williams and C. J.
Tabin, unpublished data).
3995
Journal of Cell Science 112, 3995-4005 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0801
The contractile vacuole network of Dictyostelium as a distinct organelle: its
dynamics visualized by a GFP marker protein
Daniela Gabriel, Ulrike Hacker, Jana Köhler, Annette Müller-Taubenberger, Jean-Marc Schwartz,
Monika Westphal and Günther Gerisch*
Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany
*Author for correspondence (e-mail: [email protected])
Accepted 6 September; published on WWW 3 November 1999
SUMMARY
The contractile vacuole system is an osmoregulatory
organelle composed of cisternae and interconnecting ducts.
Large cisternae act as bladders that periodically fuse with
the plasma membrane, forming pores to expel water. To
visualize the entire network in vivo and to identify
constituents of the vacuolar complex in cell fractions, we
introduced a specific marker into Dictyostelium cells, GFPtagged dajumin. The C-terminal, GFP-tagged region of this
transmembrane protein is responsible for sorting to the
contractile vacuole complex. Dajumin-GFP negligibly
associates with the plasma membrane, indicating its
retention during discharge of the bladder. Fluorescent
labeled cell-surface constituents are efficiently internalized
by endocytosis, while no significant cycling through the
contractile vacuole is observed. Endosomes loaded with
yeast particles or a fluid-phase marker indicate sharp
separation of the endocytic pathway from the contractile
vacuole compartment. Even after dispersion of the
contractile vacuole system during mitosis, dajumin-GFP
distinguishes the vesicles from endosomes, and visualizes
post-mitotic re-organization of the network around the
nucleus. Highly discriminative sorting and membrane
fusion mechanisms are proposed to account for the sharp
separation of the contractile vacuole and endosomal
compartments. Evidence for a similar compartment in
other eukaryotic cells is discussed.
INTRODUCTION
In order to distinguish the endosomal and CV compartments
in living cells, specific markers are required. Endosomes can
be loaded with fluorescent dextran (Cardelli et al., 1989; Aubry
et al., 1993; Hacker et al., 1997), and late endosomes are
selectively decorated by GFP-tagged vacuolin B (Jenne et al.,
1998). For the CV system, two markers applicable in vivo have
been described, the styryl dye FM4-64 and GFP-tagged
drainin. The fluorescent dye transiently highlights the bladder
but does not resolve the ducts, and it gradually occupies also
other internal membranes (Heuser et al., 1993). Drainin is a
peripheral membrane protein that is specifically involved in
discharge of the bladder (Becker et al., 1999). In accord with
this function, GFP-tagged drainin decorates the bladder of the
CV system.
Since drainin-GFP does not significantly label the ducts of
the CV system, no specific and stable marker is available for
the labeling of the entire CV network in vivo. We fortuitously
discovered an appropriate marker in an attempt to supply a celladhesion molecule, the contact site A (csA) glycoprotein, with
a GFP tag. CsA has been the prototype of proteins modified by
a ceramide-based phospholipid anchor (Stadler et al., 1989). A
previous study showed that this anchor is not essential for the
protein’s function in cell adhesion, but is responsible for
excluding the protein from a pathway of internalization and
In addition to the endoplasmic reticulum and the Golgi
apparatus, Dictyostelium cells contain two major vesicle
systems with distinct functions: the endosomal system and the
contractile vacuole (CV) network. Both these systems are
connected to the plasma membrane. The endosomal pathway
of nutrition begins with the uptake of particles by phagocytosis
or, in mutated strains, by the ingestion of fluid by
macropinocytosis (Hacker et al., 1997). This pathway ends
with exocytosis after a trafficking and membrane processing
period of about 90 minutes, which means that plasma
membrane and endosomal membranes are interconvertible.
The CV system consists of ducts, cisternae and bladders
(Heuser et al., 1993), and is transiently connected with the
plasma membrane by pores through which the bladders expel
water. The CV system acts primarily as an osmoregulatory
organelle, but is also involved in Ca2+ regulation (Moniakis et
al., 1999). Endosomes and contractile vacuoles share two
proteins within or at their membranes, the vacuolar H+-ATPase
(Temesvari et al., 1996) and rabD, a rab4-like small GTPase
(Bush et al., 1994). These common markers suggested that the
two compartments are connected to each other by fusion of
their membranes (Bush et al., 1996).
Key words: Endosome, Green fluorescent protein, Membrane fusion,
Organelle motility, Osmoregulation
3996 D. Gabriel and others
subsequent degradation (Barth et al., 1994). Therefore, without
impairing the activity of csA as a cell-adhesion molecule, the
lipid anchor could be replaced by the transmembrane domain
and cytoplasmic tail of another protein in order to tag the C
terminus of the protein with GFP. The other protein used to
construct a transmembrane chimera has provisionally been
designated as P29F8, the number of a cDNA clone comprising
its full-length coding region (Barth et al., 1994). We refer here
to P29F8 as dajumin, combining the initials of those who
contributed to its discovery and analysis.
When the C terminus of dajumin was tagged with GFP, the
fusion protein unexpectedly localized to the membranes of the
CV system rather than to the cell surface. With this observation
as a starting point, we studied the CV network during its
activity in vivo. The CV system is known for rapid changes in
its organization that accompany each cycle of activity. In
Fig. 1. Dajumin constructs (A) and dynamics of the CV system visualized by these GFP fusion proteins (B to D). (A) Diagram of the
csA/dajumin-GFP chimera and of dajumin-GFP. Numbers are amino-acid residues of the csA (yellow), dajumin (red), and GFP (black)
sequences, respectively. Black dots indicate potential N-glycosylation sites, regions in darker colour represent serine and threonine rich
sequence stretches, which are putative targets of O-glycosylation. Hydrophobic sequences of N-terminal leaders and of the transmembrane
domain are indicated in brown. Accession numbers are X04004 for the csA sequence and Q04286 for the dajumin sequence (referred to as
gp100 in the database). (B) A cell expressing the csA/dajumin-GFP chimera, (C,D) cells expressing dajumin-GFP. For B to D, confocal scans
of GFP fluorescence are superimposed in green to phase-contrast images in blue. The planes of focus were adjusted either close to the bottom
surface of the cells (B,C), or slightly beyond the layer of connecting ducts to illustrate the bladders in optical cross-sections (D). Time is
indicated in seconds. All three image series show asynchronous filling and discharge of bladders. During filling phases, ducts of the CV
complex expand into irregularly shaped ventricles (10 and 20 second frames in C), which succesively merge into larger vacuoles that finally
give rise to the bladder (30 to 50 second frames). Contraction of the bladder is accompanied by rosette-like thickening of its surface, suggesting
folding of the membrane (80 second frame in C). The clustered remnants of the bladder membrane rapidly develop into a network of ducts (100
second frame in C). In D, vacuoles merging along a duct are indicated by arrowheads. For B to D, the cells were attached to glass coverslips
and overlaid with agar to limit their thickness. The cell in C has been fed with yeast particles, which are located out of focus in the upper region
of the cell. Bar, 10 µm.
Contractile vacuole GFP marker 3997
the absence of any label, reflection interference contrast
microscopy visualized elements of the system that are in close
proximity to the plasma membrane (Gingell et al., 1982). This
technique provided evidence that ducts and bladders are
interconvertible (Heuser et al., 1993).
Dajumin-GFP constructs are stably integrated into the CV
membranes. They proved to be highly specific not only to the
network of ducts and bladders in interphase cells, but also to
the fragmented network of cells in mitosis (Zhu et al., 1993).
Using these constructs as markers of the CV complex, we
address the following questions. (1) Is there a connection
between the CV network and the endosomal system; (2) is
there an exchange of membrane constituents between the
bladder and the plasma membrane during discharge; (3) which
are the steps in differentiation of a bladder out of the network
of ducts; (4) how is the fragmented CV network reorganized
after mitosis?
MATERIALS AND METHODS
Construction of vectors and transformation of
Dictyostelium cells
To construct the GFP-tagged csA/dajumin chimera (Fig. 1A), the
csA/dajumin construct was isolated from the pDEV/CP fusion vector
(Barth et al., 1994). For the full-length dajumin sequence, a genomic
clone was amplified by PCR. These fragments were cloned into the
EcoRI site of pDEX RH (Faix et al., 1992) in frame with the GFP
S65T sequence (Heim and Tsien, 1996), which was inserted into the
HindIII site of the vector. The hexapeptide linker EFKKLK between
dajumin and GFP resulted from the cloning procedure. The amplified
dajumin sequence and the correct fusion of the csA, dajumin, and GFP
coding sequences were confirmed by custom sequencing (Toplab,
Martinsried).
Cells transformed by electroporation were selected for G418
resistance using 20 µg/ml of Geneticin (Sigma) and subsequently
cloned by spreading onto SM agar plates with Klebsiella aerogenes.
G418-resistant clones were screened for the expression of vectorencoded proteins by microscopic assessment of GFP fluorescence in
growth-phase cells.
Strains and culture conditions
The D. discoideum parent strain of transformants was AX2 clone 214.
For dajumin-GFP as a CV marker, two independent transformant
clones, HG1752 and HG1753, were used with indistinguishable
results. In each clone, the dajumin-GFP construct was brilliantly
expressed in almost all of the cells. For the expression of
csA/dajumin-GFP, clone HG1764 was used. All clones tested
expressed this chimera only in a small percentage of cells. The GFPα-tubulin expressing strain was HG1668 (Neujahr et al., 1998), the
control strain producing free GFP was HG1694. For Fig. 3, the csAnull mutant HG1287 was used as a control, HT-C1 as a csA expressing
and HT-CP8 as a csA/dajumin expressing transformant of this mutant
(Barth et al., 1994).
Cells were cultivated in plastic Petri dishes in liquid nutrient
medium, washed in 17 mM K/Na-phosphate buffer, pH 6.0, to
remove fluorescent compounds from the medium, and
subjected to confocal microscopy.
Immunofluorescence labeling and immunoblotting
For antibody labeling, cells were washed twice in 17 mM Na/Kphosphate buffer, pH 6.0, and allowed to adhere to glass coverslips
for 20 minutes. The cells were then fixed with picric
acid/paraformaldehyde (Humbel and Biegelmann, 1992), postfixed
Fig. 2. Three-dimensional reconstruction of the CV complex in a
living cell expressing dajumin-GFP. The cell adhered to a glass
surface and is viewed in stereo images towards the bottom (A) and
towards its free top surface (B). The images showing ducts and
bladders are constructed from series of confocal sections scanned at
intervals of 0.5 µm in the z-direction, and the time interval between
two scans was 5 seconds. Fluorescence intensities are colour-coded
from blue (low) to yellow (high). For reasons of data acquisition, the
fluorescence intensities were lower in the upper than in the bottom
half of the cell, so that colour codes are slightly different, as
indicated on the linear intensity scales. Frame width corresponds to
26 µm.
with 70% ethanol, and incubated overnight with mAb 24-210-2
against O-linked oligosaccharides or mAb 41-71-21 against the csA
protein (Bertholdt et al., 1985), followed by 2 hours of incubation with
TRITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).
Images were taken using an Axiophot 2 microscope (Zeiss) with a
×100/1.3 Neofluar objective and a cooled SensiCam CCD camera
(PCO Computer Optics).
For immunoblotting, proteins of total cell homogenate from 1×106
cells per lane were resolved by SDS-PAGE in 10% gels, blotted and
immunolabeled either with mAb 264-449-2 against GFP (a gift from
Markus Maniak), mAb 24-210-2 against O-linked carbohydrate
epitopes or mAb 33-294-17 recognizing the csA protein moiety
(Bertholdt et al., 1985). For detection of the first antibodies, goat antimouse IgG conjugated with alkaline phosphatase was used (Jackson
ImmunoResearch).
In vivo microscopy and fluorescence imaging
For covalent labeling of plasma membrane proteins in vivo, HG1752
cells were washed twice in 17 mM Na/K-phosphate buffer, pH 8.0,
and resuspended in a solution of 50 µM of the monofunctional NHS
ester of the fluorescent dye Cy 3.5 (Amersham Pharmacia Biotech) in
the same buffer. The cells were rotated slowly in the dark at 4°C for
30 minutes, washed twice in 17 mM K/Na-phosphate buffer, pH 8.0,
and once in the same buffer adjusted to pH 6.0. Cells were warmed
up to room temperature, allowed to adhere to glass coverslips, and
subjected to confocal imaging with or without agar overlay (Yumura
et al., 1984).
3998 D. Gabriel and others
Fig. 3. Residence of the non GFP-tagged csA/dajumin chimera on the cell surface in addition to its intracellular localization. Fixed cells were
labeled with mAb 71 that specifically recognizes the csA protein moiety in the normal, phospholipid anchored cell-adhesion protein (A) and
also the N-terminal csA portion in the chimera (C-F). Both constructs were expressed in csA-null cells. Untransformed csA-null cells were used
as a control for specificity of the antibody (B). The csA adhesion protein showed its normal localization on the cell surface with an apparent
accumulation at areas of intercellular contact (A). The chimera decorated, in addition to intracellular vesicles (arrowhead in D), also the entire
cell surface (C,D). In accord with the activity of the chimera in cell adhesion, it became accumulated at areas of intercellular contact (E) and on
thin tethers connecting detached cells (arrowheads in F). For immunolabeling, growth-phase cells were used, in which the csA constructs were
expressed under control of the actin15 promoter. Bars, 10 µm; the bar on the left applies to all panels except for the right one.
For the labeling of endocytic vesicles with a fluid-phase marker,
adherent cells were incubated with 17 mM K/Na-phosphate buffer,
pH 6.0, containing 2 mg/ml of TRITC-dextran (Sigma). To label
phagocytic vesicles, cells were incubated with heat-killed yeast
particles (Sigma) in liquid nutrient medium for 1 hour. Thereafter, the
medium was replaced by 17 mM K/Na-phosphate buffer, pH 6.0, and
the cells were subjected to agar overlay. Confocal imaging was
performed with an LSM 410 laser scanning microscope (Zeiss)
equipped with a ×100/1.3 Plan-Neofluar objective. 3-D images were
constructed using AVS software (Advanced Visual Systems) as
described by Neujahr et al. (1997).
Cell fractionation
Cells were suspended in 5 mM Na-glycinate buffer, pH 8.5,
containing 100 mM sucrose (Padh et al., 1991), and lysed by passing
through two layers of Nucleopore track etch membranes, pore size
8 µm (Corning). The lysate was layered on top of a gradient of 28,
40, and 60% sucrose in the Na-glycinate buffer, and centrifuged for
1 hour at 40,000 rpm in a Sorvall SW 41 rotor.
RESULTS
A selective marker of the contractile vacuole
network
With the intention of connecting the csA cell adhesion protein
through a transmembrane domain with GFP, a csA/dajuminGFP chimera was constructed which turned out to label the
bladders as well as the ducts of the CV system (Fig. 1A). The
series of confocal fluorescence images shown in Fig. 1B
illustrates the periodic changes in the pattern of the network
that are associated with cycles of vacuole activity.
To explore the possibility that the dajumin portion is
responsible for the unpredicted localization of the GFP-tagged
csA/dajumin chimera to the CV complex, we tagged fulllength dajumin with GFP (Fig. 1A). In a plane close to
the substrate surface, the fusion protein visualized the
characteristic network of ducts and cisternae of the CV
complex, in which larger cisternae act as bladders (Fig. 1C).
An optical cross-section through the cisternae slightly beyond
the network of ducts showed not only discharge of a bladder
but also the merging of cisternae along connecting ducts
(Fig. 1D).
Almost all the cells in a population expressed the dajuminGFP, and its fluorescence was brilliant. This construct could be
used, therefore, to visualize the three-dimensional organization
of the contractile vacuole complex. Serial sectioning of an
entire cell by a set of confocal planes revealed extension of the
CV network around the entire cell body, although it is most
densely knit at the substrate-attached area of the cell surface
(Fig. 2). Similarly, bladders are most often formed in the
bottom half, but also close to the upper surface of the cell.
Requirements for protein targeting to the CV
complex
The restriction of the csA/dajumin-GFP fusion protein to the
CV network was unpredicted since csA/dajumin acts as a celladhesion protein, which means that the protein is exposed
on the cell surface (Barth et al., 1994). To confirm this
observation, we immunolabeled cells expressing either normal,
lipid-anchored csA or csA/dajumin without the GFP tag (Fig.
3). Using an anti-csA antibody, the chimera was not only
localized to intracellular vesicles, some of them recognizable
as contractile vacuoles (arrowhead in Fig. 3D), but also to the
cell surface (Fig. 3C-F). Similar to the phospholipid-anchored
csA protein in Fig. 3A, the chimera accumulated at areas of
intercellular adhesion (Fig. 3E) and remained there on tethers
connecting two separated cells (Fig. 3F). In accord with the
location of the csA/dajumin chimera on the cell surface, cells
expressing this protein strongly tended to agglutinate. One has
to conclude from these data that the fusion of GFP to the C
terminus of csA/dajumin supports retention of the protein
within the CV system.
Contractile vacuole GFP marker 3999
developmentally regulated proteins were undetectable in
growth-phase cells, and both were clearly recognized after 6
hours of starvation in aggregating wild-type cells (Fig. 4A and
B). The dajumin-GFP fusion protein, which was constitutively
expressed under control of the actin15 promoter, was
recognized by mAb 210 in growth-phase as well as in
aggregating cells, and was also detected by an anti-GFP
antibody (Fig. 4A and C). These results indicate that the GFPfusion protein, like normal dajumin, is passaged through the
Golgi apparatus where it is O-glycosylated before being sorted
to the CV system.
In Fig. 4C a protein fragment with an apparent molecular
mass of 36 kDa is labeled with anti-GFP antibody. This
fragment is about 8 kDa larger than free GFP (Fig. 4D), and
thus appears to be a product of proteolytic cleavage at a
lumenal site of dajumin.
Fig. 4. O-glycosylation of dajumin indicates that the contractile
vacuole system is a post-Golgi compartment. Immunoblots were
labeled for an epitope on O-glycosylated proteins (A), for the csA
protein (B), or for GFP (C,D). Cells were harvested either at growth
phase (t0) or at the aggregation stage after 6 hours of starvation (t6).
Total cellular proteins of wild-type AX2, of a dajumin-GFP
expressing transformant of this strain, or of a strain producing free
GFP were loaded as indicated. In A, three bands of O-glycosylated
proteins are recognized by the antibody. Two of the labeled
glycoproteins are developmentally regulated and present in both
wild-type and dajumin-GFP expressing cells. The 80 kDa band
coincides with the position of the csA glycoprotein, as shown in B,
the other represents dajumin (Müller-Taubenberger, 1989). The third
band in the 127 kDa position is specific to the transformant and
present in both developmental stages. Identity with the dajumin-GFP
fusion protein is confirmed by labeling with anti-GFP antibody in C.
In addition, this antibody labels a faint band in the 36 kDa position.
The apparent molecular mass of this polypeptide is 8 kDa larger than
that of free GFP (D), suggesting that the 36 kDa band represents an
N-terminally truncated fragment of the fusion protein.
To provide evidence that dajumin enters the CV complex
after being passaged through the Golgi apparatus, modification
of the protein by O-glycosylation was examined. Among a
series of monoclonal antibodies that recognize exclusively
epitopes on O-glycosylated proteins (Bertholdt et al., 1985;
Hohmann et al., 1987), mAb 210 was selected because the
epitopes for this antibody were restricted to two Oglycosylated proteins, csA and dajumin. Both these
Cell-surface constituents are not significantly
internalized into the contractile vacuole system
The dajumin-GFP clusters left after discharge of a bladder are
often flat and spread beneath the cell boundary, so one might
falsely assume them to be incorporated into the plasma
membrane. However, live observation revealed that the clusters
can be refilled, which means they represent folded membranes
underlying the cell surface rather than protein that has diffused
from the vacuole into the plasma membrane.
Since the GFP-tag may prevent not only the csA/dajumin
chimera but also full-length dajumin from being incorporated
into the plasma membrane, GFP fusion proteins may not be
representative for the exchange of membrane proteins between
the cell surface and the CV system. To explore the possibility
of an exchange during the period of discharge of the bladder,
when the plasma membrane is connected to the CV system
through a pore, the fluorescent dye Cy 3.5 was covalently and
unselectively conjugated to constituents of the cell surface.
As a control, Fig. 5A shows the internalization of a cellsurface area during macropinocytosis. This entry into the
endosomal pathway is characterized by the budding of a fluidfilled vesicle from the plasma membrane. The Cy 3.5 label
reliably identified the internalized membrane during and after
the uptake process and, at a later stage, decorated also
membranes folded within the lumen of the vesicle (410 second
frame of Fig. 5A).
In Fig. 5B, the cell-surface label is superimposed to the
dajumin-GFP fluorescence. Several vacuoles are refilled during
the period of recording. The sequence of shape changes is best
represented by the vacuole on the left (arrowheads in the 0 and
280 second frames of Fig. 5B). The filling bladder spreads
initially beneath the cell surface, most obviously in the 130
second frame of Fig. 5B. Subsequently, the area of contact with
the cell cortex is reduced, so that the vacuole rounds up before
discharge of its contents (160 and 330 second frames of Fig.
5B). No overlap in the vacuole and cell-surface label has been
recognizable during the periods of discharge and refilling of
the bladders, indicating that translocation of proteins from the
plasma membrane to the vacuolar membrane during a cycle of
CV activity is negligible.
The contractile vacuole network is separated from
the endosomal compartment
After a period of more than 10 minutes of internalization into
4000 D. Gabriel and others
Fig. 5. Plasma membrane is efficiently internalized by macropinocytosis but not by contractile vacuole activity. Plasma membrane
internalization by endocytosis (A) is compared with filling of the contractile vacuole (B). Time at which confocal images were scanned is
indicated in seconds. (A) Internalization of plasma membrane by endocytosis. The surface of this cell was fluorescent-labeled by conjugation
with Cy 3.5 (red). At the upper right corner, fluid is taken up into a macropinosome. After internalization, the cell-surface label remains linked
to the membrane of the endosome. (B) A surface-labeled cell as in A (red) expressing dajumin-GFP (green), showing cycles of CV activity.
Superimposition of the two labels would result in yellow color. Refilling phases and discharge of vacuoles are indicated by open and filled
arrowheads, respectively. In none of these vacuoles is plasma membrane label detectably incorporated into the vacuole. The vacuole on the left
shows from 0 to 160 seconds characteristic shape changes during the filling phase: first it spanned along the cell border (130 seconds). At this
stage vacuoles are connected to tubes (140 seconds). Before discharge, the vacuole rounds up, remaining in contact with the cell cortex only at
the site of subsequent discharge. Bar, 10 µm.
endosomes, the membrane label of Cy 3.5 fades out either
by bleaching or proteolytic degradation. Therefore, the
experiments shown in Fig. 5 do not rule out an exchange
between the endosomal and the CV system at a later stage of
endosomal processing. In order to probe for an exchange, we
have used two non-digestible markers of the endosomal
pathway, boiled yeast particles and TRITC-dextran
(Rauchenberger et al., 1997). Fig. 6A depicts phase-contrast
images and confocal sections through two cells loaded with
yeast. The numerous ingested yeast particles visualized by
phase contrast (in blue) occupied much of the intracellular
space in these cells. The CV network (in green) was well
developed in a particle-free plane close to the substrate surface,
and otherwise was squeezed in form of ducts and vesicles into
the space between phagosomes filled with the yeast particles.
In cells not fed with yeast particles, endosomes are
recognized by phase contrast as fluid-filled vesicles of variable
sizes, which are not labeled with dajumin-GFP. In the example
of Fig. 6B, a tube of the CV complex surrounds in a U-turn
one of the larger unlabeled vesicles, suggesting close contact
but no fusion of the two vesicle systems. For unequivocal
distinction of endosomes from structures of the CV complex,
cells were loaded with the fluid-phase marker TRITC-dextran.
Since the average residence time between endocytosis and
exocytosis of a fluid phase marker is about 60 minutes in
Dictyostelium cells (Jenne et al., 1998), bathing the cells for at
least 90 minutes in a solution of TRITC-dextran visualizes the
lumen of endosomes at all stages of their pathway. To provide
optimal conditions for optical inspection, the cells were
compressed between a glass and agar surface. In the doublelabeled cells we never observed merging of the two labels: the
endosomal fluid-phase marker did not leak into the CV
network, nor did we recognize an enrichment of dajumin-GFP
at the site of endosomal membranes. Analysis of doublelabeled cells by series of confocal sections revealed that even
ducts of the CV system and endosomes residing in close
contact to each other did not merge into one hybrid vesicle
filled with TRITC-dextran and decorated on its surface with
GFP-dajumin (Fig. 6C).
Since no TRITC-dextran was detectable in vesicles labeled
with dajumin-GFP, it appeared unlikely that external fluid
enters the CV system during discharge or refilling of a bladder.
The lack of significant fluid uptake is explicitly demonstrated
in Fig. 6D for the two bladders marked by arrowheads. These
data indicate that during discharge of a bladder the outsidedirected flow surpasses the rate of diffusion of macromolecular
dextran into the opposite direction, and they confirm that the
bladder is closed against the environment during the filling
period. As a bonus, the sequence of Fig. 6D shows in the 490
and 500 second frames the fusion of an expanded bladder with
a second one that is just starting to refill.
To establish that constituents of the CV complex are
separable from endosomes, double-labeled cells were lysed
and particles fractionated in a sucrose gradient according to the
method of Padh et al. (1991). The endosomes loaded with
TRITC-dextran were recovered on the bottom of the gradient
Contractile vacuole GFP marker 4001
Fig. 6. The contractile
vacuole network is separated
from endosomes. Cells
expressing dajumin-GFP were
either fed with yeast particles
(A), left untreated (B), or
incubated with the fluid-phase
marker TRITC-dextran (C,
D). (A) The CV network is
shown in confocal sections
through two cells. The
fluorescent label in green is
superimposed to phasecontrast images in blue
visualizing the particles. The
cells had been pre-incubated
for 80 minutes with the
particles, and were
subsequently overlaid with
agar for imaging. Numbers
indicate distances in µm of
the optical sections from the
bottom surface of the cells.
The CV network is
concentrated on the bottom
surface and extends from
there into the clefts of
cytoplasm that are left free of
phagosomes. (B) Confocal
section through a cell as in A,
but with empty, i.e. fluid-filled
endosomes, showing a tube of
the CV complex bent around
one of these endosomes.
(C) A cell expressing
dajumin-GFP bathed in
TRITC-dextran was scanned
in different confocal planes to
visualize the spatial
relationship of contractile vacuoles (green) and endosomes (red). Upper numbers indicate distances from the bottom surface of the cell, lower
numbers time in seconds. Cell shape has changed during the period of scanning; the largest endosome is the same in all frames. Separation of
contractile vacuoles from endosomes is most clearly recognizable in the 230 second frame, where the large endosome and a contractile vacuole
are located in the plane of focus. The yellow color in the 110 second frame is due to the location of the large endosome just above the plane of
focus. Similarly, yellow color in the 440 and 780 second frames identifies ducts of the CV network outside of this endosome. (D) Discharge and
refilling of two bladders is not accompanied by detectable uptake of the fluid-phase marker into the CV complex. Open and filled arrowheads
indicate filled and discharged states of the vacuoles, respectively. For C and D, cells were incubated for 1 hour in buffer containing TRITCdextran, and were subsequently overlaid for 30 to 45 minutes with agar soaked with TRITC-dextran to label endosomes in all stages of
intracellular trafficking. Bar, 10 µm.
in the 60% sucrose fraction, in coincidence with fractionation
of the lysosomal marker acid phosphatase (Barth et al., 1994).
GFP labeled structures microscopically identifiable as ducts
and bladders were separated from the endosomes. They peaked
in the 28% sucrose fraction in accord with acidosomes (Nolta
et al., 1991), which are thought to be fragments of the CV
complex (Heuser et al., 1993; Bush et al., 1994).
Periodic activity and specific fusion of dispersed CV
vesicles in mitotic cells
By the labeling of fixed cells with calmodulin antibodies, Zhu
and Clarke (1992) have shown that the CV network is
fragmented during mitosis. In accord, many small contracting
vacuoles are seen in time-lapse movies to be dispersed
throughover the periphery of dividing cells (Gerisch, 1964).
The disconnected vesicles in mitotic cells make it possible to
study the periodic activity of a minimal unit of the CV system
apart from its integration into the circumferential network of
interphase cells (Fig. 7A). A typical sequence of bladder-duct
interconversion in a fragment of the CV complex is shown in
Fig. 7B. In the filling phase the bladder is connected with short
ducts that have blind ends. These pieces of ducts disappear or
are disconnected when the bladder starts to contract. The
membranes of the bladder collapse during contraction into a
fluorescent cluster unresolvable by optical means. The high
local fluorescence intensity in this cluster indicates that the
membranes are strongly folded, enveloping a minimum of
lumenal space. Refilling is initiated by the radial outgrowth of
tubules from the cluster. Subsequently, the lumen of the
complex dilates locally into ventricles, at the site of the
previous bladder and also within the elongating tubules. Finally
these ventricles merge, giving rise to an expanding vacuole,
4002 D. Gabriel and others
thus reconstituting after a period of 75 seconds the minute
complex of a bladder with short connected ducts.
At the end of mitosis, the CV fragments start to re-assemble
around each of the daughter nuclei (Fig. 7C). The separate CV
vesicles consecutively fuse with each other in a variable
pattern. In comparing all records of mitotic cells we have
collected, we could not find a pre-fixed site of post-mitotic CV
assembly. This is clearly different from re-assembly of the
Golgi apparatus. Golgi vesicles assort along microtubules and
are transported towards their minus end, where the Golgi
apparatus is reconstituted close to the centrosome (Schneider
et al., 1999). In contrast, CV vesicles cluster and fuse at any
side of the nuclei, including the zone opposite to centrosome
position. These data exclude the centrosome as the center of
CV assembly.
In Fig. 7D the microtubule system is visualized by GFP-αtubulin in order to relate CV reassembly to the state of mitosis.
The spindle is already disassembled when cleavage of the cell
progresses: the 100 second frame of Fig. 7D represents the
same stage of cytokinesis as the 10 second frame in Fig. 7C,
where CV reassembly is just commencing. The red label in
Fig. 7D shows endosomes loaded with TRITC-dextran,
demonstrating the absence of any pattern of endosome
assembly in the course of mitotic cell division.
To establish that contractile vacuoles stay separate from
endosomes during mitosis, we labeled cells expressing
dajumin-GFP with TRITC-dextran and searched for dividing
cells. Through all stages of mitosis, the GFP-labeled vacuoles
and TRITC-dextran loaded endosomes existed as two distinct
classes of vesicles (Fig. 7E). This example has been chosen
because it demonstrates in the upper daughter cell how a CV
ring around the nucleus is generated, giving rise to the
arrangement of ducts and bladders established in the interphase
cell of Fig. 1B. When dividing cells were strongly compressed
between a glass and agar surface, the endosomes were slightly
concentrated in the mid-zone, while the CV vesicles assembled
in the polar regions (Fig. 7F).
DISCUSSION
A specific marker of the contractile vacuole complex
applicable to studies in vivo and in vitro
In this paper we have introduced a specific marker of the
contractile vacuole system of Dictyostelium cells. As a
transmembrane protein, dajumin-GFP provides a reliable label
to study the dynamics of the entire CV complex in interphase
and mitotic cells. The CV compartment illuminated by
dajumin-GFP shows the organization and variability of
unlabeled CV structures visualized by reflection interference
contrast microscopy (Heuser et al., 1993). The fluorescent
network is also coincident with electron micrographs of freezedried broken cells showing clusters and arrays of ducts and
cisternae (Heuser et al., 1993; Clarke and Heuser, 1997). In
particular, the proposed interconversion of ducts into bladders
during cycles of activity in the CV complex (Heuser et al.,
1993) is reflected in the fluorescence images obtained with
dajumin-GFP as a marker (Figs 1B and 7B).
Since the vacuolar H+-ATPase is present not only on the CV
complex but also on the endosomes, dajumin-GFP is the only
integral membrane protein available to unequivocally identify
constituents of the contractile vacuole system in cell fractions.
Potential applications of the dajumin-GFP marker include the
analysis of mutants with impaired structure and function of the
CV complex. Examples are mutants deficient in clathrin heavy
chains or drainin, and cells overexpressing dominant negative
rabD. In the clathrin mutant, bladders are rudimentary
(Ruscetti et al., 1994), in the drainin mutant they are
excessively expanded and arrested at a stage preceding
discharge (Becker et al., 1999), and in the rabD mutant the CV
complex appears to be collapsed into one patch (Bush et al.,
1996).
It has not been within the scope of this paper to investigate
the function of dajumin used here as a marker. Dajumin knockout mutants have shown that the protein is not essential for
survival of the cells (Müller-Taubenberger, 1989). Endogenous
dajumin expressed under control of its own promoter is a
strictly developmentally regulated protein (Fig. 4A). In the
growth-phase cells, which have been used throughout the
present study, no dajumin mRNA has been detected by
Northern blotting (Gerisch et al., 1985; in this reference
dajumin has been referred to as P29F8). Dajumin mRNA
accumulates during development to the aggregation stage in
response to periodic cyclic AMP signals, in parallel with a
number of other membrane proteins that serve different
functions in aggregating cells. The role of dajumin at this stage
remains to be elucidated.
Coordination of activities in the contractile vacuole
system
Our data underline previous results indicating that the CV
network is an intracellular compartment unique in its capacity
to coordinate activities in space and time by membrane fusion,
extension and vesiculation of tubules, pore formation, and
contraction (Heuser et al., 1993; Clarke and Heuser, 1997).
This is illustrated by the steps involved in discharge of the
bladder. The bladder is ready for discharge when it reaches a
certain size. Mediated through a sensing and signal
transduction mechanism that probably involves drainin, the
state of filling appears to regulate the interaction of the bladder
with the plasma membrane (Becker et al., 1999). In a first
stage, the expanding bladder is attached to the cell cortex over
a large area of its surface, as depicted in the 20 and 130 second
frames of Fig. 5B. In drainin-null mutants, largely oversized
bladders are arrested in that particular state. Actin filaments
and palisade-like arrays of spacers separate the vacuolar
membrane from the plasma membrane in drainin-deficient
cells. In wild-type cells, this state is transient and followed by
rounding up of the bladders in preparation of their discharge
(Fig. 5B). When the pore between bladder and plasma
membrane is opened, ducts have to be closed or detached from
the bladder in order to avoid discharge of fluid into the ducts
rather than the environment (John Heuser, personal
communication). This separation of the bladder from
surrounding ducts is not always perfect; sometimes we have
seen the blowing up of adjacent tubular regions during the
contraction of a bladder.
A functional differentiation between bladders and ducts is
illustrated by the preferential association of drainin with the
bladders at all stages of a contraction cycle (Becker et al.,
1999). On the other hand, our results substantiate
previous observations that ducts and bladders are readily
Contractile vacuole GFP marker 4003
Fig. 7. Activities in the dispersed CV complex during mitosis, and the mode of its reorganization. Mitotic cell division was recorded by the
confocal scanning of cells expressing either dajumin-GFP (A-C,E,F) or GFP-α-tubulin (D). The latter is used as a marker of the mitotic
apparatus in order to relate progression of mitosis to cytokinesis. GFP fluorescence in green is superimposed to phase-contrast images in blue.
Time is indicated in seconds. (B) shows one contraction-expansion cycle of a CV fragment marked by arrowhead in the 355 second frame of A.
The time scale in B is accordingly fit to that in A. Cells were gently, or in F strongly compressed by agar overlay. For D to F, endosomes were
preloaded for 1 hour with TRITC-dextran, which is shown in red, and washed in 17 mM K/Na-phosphate buffer, pH 6.0, before overlay with
agar in the same buffer. For the cell in E, the focus was repeatedly changed from a plane where the majority of endosomes and the nuclei are
located (0 and 140 second frames) and a plane closer to the substrate where the CV complex is reassembling (all other frames). Bars, 10 µm.
interconverted during filling and discharge (Heuser et al.,
1993). Locally regulated contractility is one of the
mechanisms involved in bladder to duct conversion. The
transfer of fluid from one vacuole into another, and the
differentiation of membrane clusters into ducts and bladders
during the filling period (Figs 1D, 5B and 6D) suggests the
presence of a delicately controlled system for contractility in
the CV complex. The availability of dajumin-GFP as a
specific marker makes it possible to localize unconventional
myosins or other motor proteins to the CV arrays and to
identify constituents of the cytoskeleton that anchor the
network at the cell cortex.
Protein targeting to the CV complex
Dajumin has an N-terminal hydrophobic leader sequence and
a transmembrane domain in its C-terminal half, indicating that
the protein is integrated into the membrane upon its synthesis
in the ER. O-glycosylation is a post-translational modification
in Dictyostelium as in other cells (Hohmann et al., 1987). The
presence of O-linked oligosaccharides on dajumin and its GFP
4004 D. Gabriel and others
fusion product suggests that the protein is passaged through the
Golgi apparatus on its way to the CV complex. The question
is how dajumin-GFP is distinguished from proteins sorted to
the plasma membrane or to the endosomal pathway. In the
csA/dajumin-GFP chimera the N-terminal csA portion is
localized on the lumenal side of the vacuolar membrane. Since
csA is a phospholipid anchored cell-adhesion molecule
normally kept on the cell surface (Barth et al., 1994), it is
unlikely that the csA portion targets the chimera to the CV
complex. Since free GFP is uniformly distributed in the
cytoplasm of Dictyostelium cells and slightly enriched in the
nuclear matrix, the C-terminal GFP sequence cannot be
responsible for targeting the chimera to the CV system.
Therefore, localization to the CV complex is attributed to the
dajumin fragment in the middle of the csA/dajumin-GFP
chimera. This dajumin fragment consists of the C-terminal
region of dajumin including a stretch of 18 amino-acid residues
on the lumenal side of the membrane, a trans-membrane
domain of 23 hydrophobic residues and a cytoplasmic tail of
34 residues (Fig. 1A). It will be necessary to dissect the protein
further in order to analyse the sequence requirements for
protein targeting to the CV complex.
Although the GFP moiety has no targeting capacity by itself,
it assists in restricting the fusion protein to the CV system.
Untagged csA/dajumin is detectable by antibody on the cell
surface (Fig. 3), and is active there as a cell adhesion protein
(Barth et al., 1994). Only the GFP-tagged csA/dajumin is
efficiently entrapped within the CV system (Fig. 1B). The
presence of the GFP moiety is therefore crucial for the use of
dajumin constructs as CV markers.
TRITC-dextran enter the ducts or bladders of the CV network.
We conclude that under normal conditions the CV
compartment is closed to vesicles of the endosomal pathway,
prohibiting lumenal transit from endosomes to the CV
network.
The CV complex is an integral compartment
separated from endosomes and the plasma
membrane
The results obtained with the dajumin-GFP marker indicate
that membrane flow and the exchange of contents between the
CV complex and other compartments is strictly controlled.
During discharge of the bladder, dajumin-GFP accumulates
beneath the plasma membrane in patches that remain part
of the contractile vacuole system (Figs. 5B and 6D). This
retention of the marker argues against an unselective transit of
proteins from the vacuolar membrane to the cell surface. Only
exceptionally we have found dajumin-GFP at the plasma
membrane, perhaps resulting from a slight injury of the cells.
(The label at the upper border of the cell in the 440 to 500
second frames of Fig. 6D could be due to such an effect.)
Evidence for regulated localization of a vacuolar protein has
been provided by Moniakis et al. (1999), who reported that the
Ca2+-ATPase PAT1 populates the plasma membrane in
response to high extracellular Ca2+ concentrations.
Our data provide no evidence for a collateral communication
between the endosomal compartment and the CV complex.
These results are in accord with observations indicating that
bladders decorated on their cytoplasmic surface with GFPdrainin were clearly distinguishable from endosomes filled
with the fluid-phase marker TRITC-dextran (Becker et al.,
1999). In the present study we show that the entire network of
ducts and vesicles of the CV system is separated from
endosomes, and that the same is true for the disassembled
vesicles in mitotic cells. Neither did dajumin-GFP detectably
decorate endosomes at any stage of their pathway, nor did
We thank John Heuser for intense discussions and Gerard Marriott
for recommending the Cy 3.5 membrane labeling procedure to us. The
work has been supported by the Deutsche Forschungsgemeinschaft
(SFB266/C6) and the Fonds der Chemischen Industrie.
Is there a compartment corresponding to the
contractile vacuole system in higher eukaryotes?
The sequence of the C-terminal region of dajumin, responsible
for targeting to the CV system, reveals no obvious sequence
relationships to other proteins in the database. However,
another Dictyostelium protein, drainin, which is specifically
associated with the contractile vacuole and involved in its
discharge, is the prototype of a protein family represented in
Caenorhabditis elegans and man (Becker et al., 1999). The
question is whether the drainin homologues of higher
eukaryotes serve similar functions as drainin in Dictyostelium,
and whether they are localized to a compartment as distinct
from endosomes as the CV complex. A compartment of
apparent similarity is the sub-plasmalemmal tubulocisternal
system characterized in neuroendocrine cells by Schmidt et al.
(1997). This membrane system is connected with the cell
surface by narrow channels and gives rise to synaptic-like
microvesicles, the counterpart of neuronal synaptic vesicles. It
will be intriguing to find out whether the CV complex,
previously considered to be a peculiarity of protozoa living
under conditions of low osmolarity, is indeed a specialized
version of a compartment in eukaryotic cells, connected to the
plasma membrane and functioning in the conversion of ducts
into vesicles.
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